Gene augmentation of LCA5-associated Leber congenital amaurosis ameliorates bulge region defects of the photoreceptor ciliary axoneme
Siebren Faber, Olivier Mercey, Katrin Junger, Alejandro Garanto, Helen May-Simera, Marius Ueffing, Rob W.J. Collin, Karsten Boldt, Paul Guichard, Virginie Hamel, Ronald Roepman
Siebren Faber, Olivier Mercey, Katrin Junger, Alejandro Garanto, Helen May-Simera, Marius Ueffing, Rob W.J. Collin, Karsten Boldt, Paul Guichard, Virginie Hamel, Ronald Roepman
View: Text | PDF
Research Article Cell biology Genetics

Gene augmentation of LCA5-associated Leber congenital amaurosis ameliorates bulge region defects of the photoreceptor ciliary axoneme

Abstract

Leber congenital amaurosis (LCA) is a group of inherited retinal diseases characterized by early-onset, rapid loss of photoreceptor cells. Despite the discovery of a growing number of genes associated with this disease, the molecular mechanisms of photoreceptor cell degeneration of most LCA subtypes remain poorly understood. Here, using retina-specific affinity proteomics combined with ultrastructure expansion microscopy, we reveal the structural and molecular defects underlying LCA type 5 (LCA5) with nanoscale resolution. We show that LCA5-encoded lebercilin, together with retinitis pigmentosa 1 protein (RP1) and the intraflagellar transport (IFT) proteins IFT81 and IFT88, localized at the bulge region of the photoreceptor outer segment (OS), a region crucial for OS membrane disc formation. Next, we demonstrate that mutant mice deficient in lebercilin exhibited early axonemal defects at the bulge region and the distal OS, accompanied by reduced levels of RP1 and IFT proteins, affecting membrane disc formation and presumably leading to photoreceptor death. Finally, adeno-associated virus–based LCA5 gene augmentation partially restored the bulge region, preserved OS axoneme structure and membrane disc formation, and resulted in photoreceptor cell survival. Our approach thus provides a next level of assessment of retinal (gene) therapy efficacy at the molecular level.

Authors

Siebren Faber, Olivier Mercey, Katrin Junger, Alejandro Garanto, Helen May-Simera, Marius Ueffing, Rob W.J. Collin, Karsten Boldt, Paul Guichard, Virginie Hamel, Ronald Roepman

×

Figure 4

Effect of lebercilin loss on OS formation and intraflagellar transport.

Options: View larger image (or click on image) Download as PowerPoint
Effect of lebercilin loss on OS formation and intraflagellar transport. ...
(A and B) Widefield (original magnification, 63×) images of expanded photoreceptors stained for tubulin (magenta) and rhodopsin (green) from P10 to P28 in Lca5+/gt (HET) (A) and Lca5gt/gt (HOM) (B) mice. White arrows indicate accumulation of rhodopsin above the basal body. White arrowheads indicate rhodopsin along the CC. Open white arrowheads indicate rhodopsin in vesicle-like structures. Scale bars: 500 nm. (C and D) Widefield (original magnification, 63×) images of expanded photoreceptors stained for tubulin (magenta) and IFT81 (yellow) from P10 to P28 in HET (C) and HOM (D) mice. White arrowheads indicate IFT81 localization above the basal body. Closed and open yellow arrowheads indicate IFT81 localization at the bulge region in HET and HOM, respectively. Scale bars: 500 nm. (E) Quantification of the distance of lebercilin (LCA5; orange; same values as in Figure 2G) and IFT81 bulge region (yellow) signal proximal ends to the mother centriole proximal end from P14 to P28. P10 not included, since the bulge region is not properly formed yet at this time point. Three animals per time point. Data presented as mean ± SD; n = 62–113. *P < 0.05 by Mann-Whitney test. (F and G) Impact of LCA5 loss on IFT81-normalized intensity above the basal body (F) or at the bulge region (G) from P10 to P28. P10 not included for IFT81 intensity at the bulge region, since it is not properly formed yet at this time point. Three animals per time point. Data presented as mean ± SD; n = 8–113 (F), n = 10–113 (G). ****P < 0.0001 by Mann-Whitney test. (H) Confocal U-ExM images of adult photoreceptor stained for tubulin (magenta) and IFT88 (yellow). Right panels show transversal view of the bulge region. White arrowhead indicates IFT88 localization above the basal body. Yellow arrowhead indicates IFT88 localization at the bulge region. Scale bars: 500 nm (side view), 200 nm (transversal view).